ABSTRACT For patients with head and neck cancer whose tumors are HPV negative, current therapy does not lead to sig- nificant longevity and most succumb to loco-regional recurrence of the primary tumor. We discovered that HPV(-) head and neck cancer patients fit into four distinct categories relative to their PD-L1 and CD8 status. These categories had discrete outcomes such as loco-regional recurrence and disease-specific survival, with CD8 and PDL-1 double positive patients faring worse than any other patient cohort. We asked if we can de- velop treatment strategies that can leverage the unique biology of these patient categories. We posit that pres- ence of PDL-1 either blocks infiltration of CD8+ cytotoxic T cells or leads to exhaustion of T cells that do make it to the tumor parenchyma. Postulating further, we asked if overexpression of surface PDL-1 can be used as a homing mechanism for radiosensitizing gold nanoparticles. We also asked if blocking PDL-1 could replicate the tumor response of PDL-1 negative cohorts. Finally, we considered strategies that can increase immunogenicity of tumors that lack PDL-1. In this proposal, we ask three main questions: 1) Can we use PDL-1 to home gold nanoparticles and enhance radiation specifically within tumor cells? We hypothesized that overexpression of PDL-1 may serve as the ideal homing strategy for radiosensitization. Gold nanoparticles are well described radiosensitizers that are easily targeted to tumors via antibodies. We will conjugate spheroid gold nanoparticles to ?-PDL-1 and examine their efficacy in vivo for tumor control and overall survival. 2) By what mechanism is PDL-1 blocking the activity of infiltrating cytotoxic T cells and can this mechanism be overturned? Using a syngeneic model of head and neck cancer, we will investigate if eliminating PDL-1 by genetic manipulation or an inhibitory antibody can improve tumor control following genotoxic therapy. We will further profile the T cell infiltrate from both the human tissues and the animal tumors and ask if the profile changes following PDL-1 in- hibition. The goal is to understand if PDL-1 overexpression in tumor cells is simply correlative to or causative of the systemic immune inhibition. 3) Can radiation increase immunogenicity of dying tumor cells? We present data to demonstrate that radiation with high linear transfer energy (LET), which can be achieved by particle therapy, increased immunogenic signals from tumor cells in vitro. Therefore, we will investigate if treatment with proton beams with high-LET can achieve a similar effect in vivo. Finally, as a test of a three-hit strategy, we will examine the tumor control that can be achieved by combining ?-PDL-1 tagged gold nanoparticles with high-LET proton radiation. We anticipate that this treatment will block T-cell exhaustion, enhance radiation ef- fects, and increase the immunogenicity of dying tumor cells. Our understanding of the interplay between tumor biology and the immune system suggests that new strategies will have to be developed that can merge our knowledge of nanotechnology, radiation response, and immunology into sophisticated and innovative treat- ment strategies.